Investigations of ion transport mechanisms in erythroid cells in 3 areas: Anion transport: We have proposed a model for the mechanism whereby the band 3 protein mediates anion exchange in human erythrocytes which postulates that a single transport site can oscillate between two conformations, one facing the inside of the cell and the other facing the outside. To test this model, chloride gradients will be imposed across the membrane to determine if they have the predicted effects on the inhibitory potency of chemical probes, which bind to specific functional sites of the transport system. These experiments will also reveal any intrinsic asymmetry in the orientation of the transport sites toward the inside or outside of the cell, as well as possible effects of the conformational chage on other band 3 sites and on band 3-associated lipids. Chemical probes will also be used to identify those segments of the band 3 protein which are adjacent to functional sites. The relationship of net anion flow to the anion exchange system will also be investigated, and a model for the mechanism of net anion flow will be tested. The results should improve our understanding of the biochemical mechanism of this and other protein-mediated transport processes. Band 3 biosynthesis: Because of its unusual structure, with the N-terminal end located at the cytoplasmic surface and not glycosylated, band 3 represents a challenge to current at theories for membrane protein biosynthesis. We plan to determine if the human erythroleukemic cell line K562 synthesizes band 3, by using a combination of transport kinetic, biochemical labelling, and immunological techniques, and to use this cell line to determine the mechanism by which band 3 is inserted into the membrane. If K562 cells do not synthesize band 3, mouse spleen cells will be used. Friend cell induction: Various lines of evidence suggest that changes in membrane function may be involved in the process whereby chemical inducers stimulate Friend erythroleukemic cells to produce hemoglobin. We plan to investigate the possible involvement of changes in Ca fluxes and changes in cell volume in the induction process, in order to elucidate the role of the membrane in this alteration of gene expression.